The success of the drug Enbrel® (etanercept) brought to fruition the promise of therapeutic agents modified with the constant domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and a constant domain known as “Fc”, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), Nature 337: 525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusion proteins known in the art.
TABLE 1Fc fusion with therapeutic proteinsFusionTherapeuticForm of FcpartnerimplicationsReferenceIgG1N-terminusHodgkin's disease;U.S. Pat. No. 5,480,981of CD30-Lanaplastic lymphoma;T-cell leukemiaMurine Fcγ2aIL-10anti-inflammatory;Zheng et al. (1995), J.transplant rejectionImmunol. 154: 5590-600IgG1TNFseptic shockFisher et al. (1996), N. Engl.receptorJ. Med. 334: 1697-1702; VanZee, K. et al. (1996), J.Immunol. 156: 2221-30IgG, IgA, IgM, orTNFinflammation,U.S. Pat. No. 5,808,029,IgE (excludingreceptorautoimmune disordersissued Sep. 15, 1998the first domain)IgG1CD4AIDSCapon et al. (1989), Naturereceptor337: 525-31IgG1,N-terminusanti-cancer, antiviralHarvill et al. (1995),IgG3of IL-2Immunotech. 1: 95-105IgG1C-terminusosteoarthritis;WO 97/23614, published Jul.of OPGbone density3, 1997IgG1N-terminusanti-obesityWO 98/28427, filedof leptinDec. 11, 1997Human Ig Cγ1CTLA-4autoimmune disordersLinsley (1991), J. Exp. Med.174: 561-9
A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), Science 267: 383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”).
Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26:401-24.
Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “E. coli display.” Another biological approach to screening soluble peptide mixtures uses yeast for expression and secretion. See Smith et al. (1993), Mol. Pharmacol. 43: 741-8. Hereinafter, the method of Smith et al. and related methods are referred to as “yeast-based screening.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ chemical linkage of peptides to RNA; see, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA 94: 12297-303. Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.
In the case of known bioactive peptides, rational design of peptide ligands with favorable therapeutic properties can be completed. In such an approach, one makes stepwise changes to a peptide sequence and determines the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide (e.g., solution structure). Hereinafter, these techniques are collectively referred to as “rational design.” In one such technique, one makes a series of peptides in which one replaces a single residue at a time with alanine. This technique is commonly referred to as an “alanine walk” or an “alanine scan.” When two residues (contiguous or spaced apart) are replaced, it is referred to as a “double alanine walk.” The resultant amino acid substitutions can be used alone or in combination to result in a new peptide entity with favorable therapeutic properties.
Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266-70. Hereinafter, these and related methods are referred to as “protein structural analysis.” These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.
Conceptually, one may discover peptide mimetics of any protein using phage display and the other methods mentioned above. These methods have been used for epitope mapping, for identification of critical amino acids in protein-protein interactions, and as leads for the discovery of new therapeutic agents. E.g., Cortese et al. (1996), Curr. Opin. Biotech. 7: 616-21. Peptide libraries are now being used most often in immunological studies, such as epitope mapping. Kreeger (1996), The Scientist 10(13): 19-20.
Of particular interest here is use of peptide libraries and other techniques in the discovery of pharmacologically active peptides. A number of such peptides identified in the art are summarized in Table 2. The peptides are described in the listed publications, each of which is hereby incorporated by reference. The pharmacologic activity of the peptides is described, and in many instances is followed by a shorthand term therefore in parentheses. Some of these peptides have been modified (e.g., to form C-terminally cross-linked dimers). Typically, peptide libraries were screened for binding to a receptor for a pharmacologically active protein (e.g., EPO receptor). In at least one instance (CTLA4), the peptide library was screened for binding to a monoclonal antibody.
TABLE 2Pharmacologically active peptidesForm ofBinding partner/peptideprotein of interestaPharmacologic activityReferenceintrapeptideEPO receptorEPO-mimeticWrighton et al. (1996),disulfide-Science 273: 458-63; U.S.bondedPat. No. 5,773,569, issuedJun. 30, 1998 toWrighton et al.C-terminallyEPO receptorEPO-mimeticLivnah et al. (1996),cross-linkedScience 273: 464-71;dimerWrighton et al. (1997),Nature Biotechnology 15:1261-5; Internationalpatent application WO96/40772, published Dec.19, 1996linearEPO receptorEPO-mimeticNaranda et al. (1999),Proc. Natl. Acad. Sci.USA, 96: 7569-74; WO99/47151, publishedSep. 23, 1999linear; C-c-MplTPO-mimeticCwirla et al. (1997)terminallyScience 276: 1696-9; U.S.cross-linkedPat. No. 5,869,451, issueddimerFeb. 9, 1999; WO 00/24770,published May 4, 2000;U.S. patent application Ser. No.2003/0176352, publishedSep. 18, 2003; WO03/031589, publishedApr. 17, 2003disulfide-stimulation ofPaukovits et al. (1984),linked dimerhematopoiesisHoppe-Seylers Z.(“G-CSF-mimetic”)Physiol. Chem. 365: 303-11;Laerum et al. (1988),Exp. Hemat. 16: 274-80alkylene-G-CSF-mimeticBhatnagar et al. (1996), J.linked dimerMed. Chem. 39: 3814-9;Cuthbertson et al. (1997),J. Med. Chem. 40: 2876-82;King et al. (1991),Exp. Hematol. 19: 481;King et al. (1995), Blood86 (Suppl. 1): 309alinearIL-1 receptorinflammatory andU.S. Pat. No. 5,608,035;autoimmune diseasesU.S. Pat. No. 5,786,331;(“IL-1 antagonist” orU.S. Pat. No. 5,880,096;“IL-1ra-mimetic”)Yanofsky et al. (1996),Proc. Natl. Acad. Sci. 93:7381-6; Akeson et al.(1996), J. Biol. Chem. 271:30517-23; Wiekzorek etal. (1997), Pol. J.Pharmacol. 49: 107-17;Yanofsky (1996), PNAs,93: 7381-7386.linearFacteur thymiquestimulation ofInagaki-Ohara et al.serique (FTS)lymphocytes(1996), Cellular(“FTS-mimetic”)Immunol. 171: 30-40;Yoshida (1984), Int. J.Immunopharmacol,6: 141-6.intrapeptideCTLA4 MAbCTLA4-mimeticFukumoto et al. (1998),disulfideNature Biotech. 16: 267-70bondedexocyclicTNF-α receptorTNF-α antagonistTakasaki et al. (1997),Nature Biotech. 15: 1266-70;WO 98/53842,published Dec. 3, 1998linearTNF-α receptorTNF-α antagonistChirinos-Rojas ( ), J.Imm., 5621-5626.intrapeptideC3binhibition ofSahu et al. (1996), J.disulfidecomplement activation;Immunol. 157: 884-91;bondedautoimmune diseasesMorikis et al. (1998),(“C3b-antagonist”)Protein Sci. 7: 619-27linearvinculincell adhesionAdey et al. (1997),processes-cell growth,Biochem. J. 324: 523-8differentiation, woundhealing, tumormetastasis (“vinculinbinding”)linearC4 binding proteinanti-thromboticLinse et al. (1997), J. Biol.(C4BP)Chem. 272: 14658-65linearurokinase receptorprocesses associatedGoodson et al. (1994),with urokinaseProc. Natl. Acad. Sci. 91:interaction with its7129-33; Internationalreceptor (e.g.,application WOangiogenesis, tumor97/35969, publishedcell invasion andOct. 2, 1997metastasis); (“UKRantagonist”)linearMdm2, Hdm2Inhibition ofPicksley et al. (1994),inactivation of p53Oncogene 9: 2523-9;mediated by Mdm2 orBottger et al. (1997) J.hdm2; anti-tumorMol. Biol. 269: 744-56;(“Mdm/hdmBottger et al. (1996),antagonist”)Oncogene 13: 2141-7linearp21WAF1anti-tumor byBall et al. (1997), Curr.mimicking the activityBiol. 7: 71-80of p21WAF1linearfarnesyl transferaseanti-cancer byGibbs et al. (1994), Cellpreventing activation77: 175-178of ras oncogenelinearRas effector domainanti-cancer byMoodie et al. (1994),inhibiting biologicalTrends Genet 10: 44-48function of the rasRodriguez et al. (1994),oncogeneNature 370: 527-532linearSH2/SH3 domainsanti-cancer byPawson et al (1993),inhibiting tumorCurr. Biol. 3: 434-432growth with activatedYu et al. (1994), Celltyrosine kinases;76: 933-945; Rickles et al.treatment of SH3-(1994), EMBO J. 13: 5598-5604;mediated disease statesSparks et al. (1994),(“SH3 antagonist”)J. Biol. Chem. 269: 23853-6;Sparks et al. (1996),Proc. Natl. Acad. Sci. 93:1540-4; U.S. Pat. No.5,886,150, issued Mar.23, 1999; U.S. Pat. No.5,888,763, issued Mar.30, 1999linearp16INK4anti-cancer byFåhraeus et al. (1996),mimicking activity ofCurr. Biol. 6: 84-91p16; e.g., inhibitingcyclin D-Cdk complex(“p16-mimetic”)linearSrc, Lyninhibition of Mast cellStauffer et al. (1997),activation, IgE-relatedBiochem. 36: 9388-94conditions, type Ihypersensitivity(“Mast cell antagonist”)linearMast cell proteasetreatment ofInternational applicationinflammatory disordersWO 98/33812, publishedmediated by release ofAug. 6, 1998tryptase-6(“Mast cell proteaseinhibitors”)linearHBV core antigentreatment of HBV viralDyson & Muray (1995),(HBcAg)infections (“anti-HBV”)Proc. Natl. Acad. Sci. 92:2194-8linearselectinsneutrophil adhesion;Martens et al. (1995), J.inflammatory diseasesBiol. Chem. 270: 21129-36;(“selectin antagonist”)European patentapplication EP 0 714 912,published Jun. 5, 1996linear,calmodulincalmodulin antagonistPierce et al. (1995),cyclizedMolec. Diversity 1: 259-65;Dedman et al. (1993),J. Biol. Chem. 268: 23025-30;Adey & Kay (1996),Gene 169: 133-4linear,integrinstumor-homing;Internationalcyclized-treatment forapplications WOconditions related to95/14714, publishedintegrin-mediatedJun. 1, 1995; WOcellular events,97/08203, publishedincluding plateletMar. 6, 1997; WOaggregation,98/10795, publishedthrombosis, woundMar. 19, 1998; WOhealing, osteoporosis,99/24462, publishedtissue repair,May 20, 1999; Kraft et al.angiogenesis (e.g., for(1999), J. Biol. Chem. 274:treatment of cancer),1979-1985and tumor invasion(“integrin-binding”)cyclic, linearfibronectin andtreatment ofWO 98/09985, publishedextracellular matrixinflammatory andMar. 12, 1998components of Tautoimmune conditionscells andmacrophageslinearsomatostatin andtreatment orEuropean patentcortistatinprevention of hormone-application 0 911 393,producing tumors,published Apr. 28, 1999acromegaly, giantism,dementia, gastric ulcer,tumor growth,inhibition of hormonesecretion, modulationof sleep or neuralactivitylinearbacterialantibiotic; septic shock;U.S. Pat. No. 5,877,151,lipopolysaccharidedisorders modulatableissued Mar. 2, 1999by CAP37linear orpardaxin, mellitinantipathogenicWO 97/31019, publishedcyclic,28 Aug. 1997including D-amino acidslinear, cyclicVIPimpotence,WO 97/40070, publishedneurodegenerativeOct. 30, 1997disorderslinearCTLscancerEP 0 770 624, publishedMay 2, 1997linearTHF-gamma2Burnstein (1988),Biochem., 27: 4066-71.linearAmylinCooper (1987), Proc.Natl. Acad. Sci., 84: 8628-32.linearAdrenomedullinKitamura (1993), BBRC,192: 553-60.cyclic, linearVEGFanti-angiogenic; cancer,Fairbrother (1998),rheumatoid arthritis,Biochem., 37: 17754-17764.diabetic retinopathy,psoriasis (“VEGFantagonist”)cyclicMMPinflammation andKoivunen (1999), Natureautoimmune disorders;Biotech., 17: 768-774.tumor growth(“MMP inhibitor”)HGH fragmenttreatment of obesityU.S. Pat. No. 5,869,452Echistatininhibition of plateletGan (1988), J. Biol.aggregationChem., 263: 19827-32.linearSLE autoantibodySLEWO 96/30057, publishedOct. 3, 1996GD1alphasuppression of tumorIshikawa et al. (1998),metastasisFEBS Lett. 441 (1): 20-4antiphospholipidendothelial cellBlank et al. (1999), Proc.beta-2-glycoprotein-activation,Natl. Acad. Sci. USA 96:I (□2GPI)antiphospholipid5164-8antibodiessyndrome (APS),thromboembolicphenomena,thrombocytopenia, andrecurrent fetal losslinearT Cell Receptor betadiabetesWO 96/11214, publishedchainApr. 18, 1996.Antiproliferative,WO 00/01402, publishedantiviralJan. 13, 2000.anti-ischemic, growthWO 99/62539, publishedhormone-liberatingDec. 9, 1999.anti-angiogenicWO 99/61476, publishedDec. 2, 1999.linearApoptosis agonist;WO 99/38526, publishedtreatment of T cell-Aug. 5, 1999.associated disorders(e.g., autoimmunediseases, viral infection,T cell leukemia, T celllymphoma)linearMHC class IItreatment ofU.S. Pat. No. 5,880,103,autoimmune diseasesissued Mar. 9, 1999.linearandrogen R, p75,proapoptotic, useful inWO 99/45944, publishedMJD, DCC,treating cancerSep. 16, 1999.huntingtinlinearvon Willebrandinhibition of Factor VIIIWO 97/41220, publishedFactor; Factor VIIIinteraction;Apr. 29, 1997.anticoagulantslinearlentivirus LLP1antimicrobialU.S. Pat. No. 5,945,507,issued Aug. 31, 1999.linearDelta-Sleepsleep disordersGraf (1986), PeptidesInducing Peptide7: 1165.linearC-Reactive Proteininflammation andBarna (1994), Cancer(CRP)cancerImmunol. Immunother.38: 38 (1994).linearSperm-ActivatinginfertilitySuzuki (1992), Comp.PeptidesBiochem. Physiol.102B: 679.linearangiotensinshematopoietic factorsLundergan (1999), J. Periodontalfor hematocytopenicRes.conditions from cancer,34(4): 223-228.AIDS, etc.linearHIV-1 gp41anti-AIDSChan (1998), Cell 93: 681-684.linearPKCinhibition of boneMoonga (1998), Exp.resorptionPhysiol. 83: 717-725.lineardefensins (HNP-1, -2,antimicrobialHarvig (1994), Methods-3, -4)Enz. 236: 160-172.linearp185HER2/neu, C-AHNP-mimetic: anti-Park (2000), Nat.erbB-2tumorBiotechnol. 18: 194-198.lineargp130IL-6 antagonistWO 99/60013, publishedNov. 25, 1999.linearcollagen, other joint,autoimmune diseasesWO 99/50282, publishedcartilage, arthritis-Oct. 7, 1999.related proteinslinearHIV-1 envelopetreatment ofWO 99/51254, publishedproteinneurologicalOct. 14, 1999.degenerative diseaseslinearIL-2autoimmune disordersWO 00/04048, published(e.g., graft rejection,Jan. 27, 2000; WOrheumatoid arthritis)00/11028, publishedMar. 2, 2000.linear, cyclicvariousinflammatoryU.S. Pat. No. 6,660,843conditions,autoimmune disease,otherslinear, cyclicAng-2inhibition ofU.S. patent application Ser. No.angiogenesis (e.g., for2003/0229023, publishedtreatment of tumor)Dec. 11, 2003; WO03/057134, publishedJul. 17, 2003; U.S.2003/0236193, publishedDec. 25, 2003NGFchronic pain, migraine,WO 04/026329,asthma, hyperactivepublished Apr. 1, 2004bladder, psoriasis,cancer, other conditionslinked to NGFmyostatinU.S. Serial No.10/742,379, filed Dec. 19,2003; PCT/US03/40781,filed Dec. 19, 2003BAFF/TALL-1B-cell mediatedU.S. 2003/0195156,autoimmune diseasespublished Oct. 16, 2003;and cancers (e.g.,WO 02/092620,lupus, B-cellpublished Nov. 21, 2002lymphoma)linearGLP-1Diabetes, metabolicsyndromeaThe protein listed in this column may be bound by the associated peptide (e.g., EPO receptor, IL-1 receptor) or mimicked by the associated peptide. The references listed for each clarify whether the molecule is bound by or mimicked by the peptides.
Peptides identified by peptide library screening were for a long time regarded simply as “leads” in development of therapeutic agents rather than as therapeutic agents themselves. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticuloendothelial system, or proteolytic degradation. Francis (1992), Focus on Growth Factors 3: 4-11. As a result, the art used the identified peptides to validate drug targets or as scaffolds for design of organic compounds that might not have been as easily or as quickly identified through chemical library screening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26:401-24; Kay et al. (1998), Drug Disc. Today 3: 370-8.
A more recent development is fusion of randomly generated peptides with the Fc domain. See U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 to Feige et al. (incorporated by reference in its entirety). Such molecules have come to be known as “peptibodies.” They include one or more peptides linked to the N-terminus, C-terminus, amino acid sidechains, or to more than one of these sites. Peptibody technology enables design of therapeutic agents that incorporate peptides that target one or more ligands or receptors, tumor-homing peptides, membrane-transporting peptides, and the like. Peptibody technology has proven useful in design of a number of such molecules, including linear and disulfide-constrained peptides, “tandem peptide multimers” (i.e., more than one peptide on a single chain of an Fc domain). See, for example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No. 2003/0195156, published Oct. 16, 2003 (corresponding to WO 02/092620, published Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003 (corresponding to WO 03/031589, published Apr. 17, 2003); U.S. Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO 00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S. Pat. App. No. 2003/0236193, published Dec. 25, 2003 (corresponding to PCT/US04/010989, filed Apr. 8, 2004); U.S. Ser. No. 10/666,480, filed Sep. 18, 2003 (corresponding to WO 04/026329, published Apr. 1, 2004), each of which is hereby incorporated by reference in its entirety. The art would benefit from further technology enabling such rational design of polypeptide therapeutic agents.